Hypoxia determines survival outcomes of bacterial infection through HIF-1alpha dependent re-programming of leukocyte metabolism.

Hypoxia and bacterial infection frequently co-exist, in both acute and chronic clinical settings, and typically result in adverse clinical outcomes. To ameliorate this morbidity, we investigated the interaction between hypoxia and the host response. In the context of acute hypoxia, both S. aureus and S. pneumoniae infections rapidly induced progressive neutrophil mediated morbidity and mortality, with associated hypothermia and cardiovascular compromise. Preconditioning animals through longer exposures to hypoxia, prior to infection, prevented these pathophysiological responses and profoundly dampened the transcriptome of circulating leukocytes. Specifically, perturbation of HIF pathway and glycolysis genes by hypoxic preconditioning was associated with reduced leukocyte glucose utilisation, resulting in systemic rescue from a global negative energy state and myocardial protection. Thus we demonstrate that hypoxia preconditions the innate immune response and determines survival outcomes following bacterial infection through suppression of HIF-1α and neutrophil metabolism. The therapeutic implications of this work are that in the context of systemic or tissue hypoxia therapies that target the host response could improve infection associated morbidity and mortality.

Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.

The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186 , in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

The rise and rise of Staphylococcus aureus: laughing in the face of granulocytes.

Recent developments in the study of host-pathogen interactions have fundamentally altered our understanding of the nature of Staphylococcus aureus infection, and previously held tenets regarding the role of the granulocyte are being cast aside. Novel mechanisms of pathogenesis are becoming evident, revealing the extent to which S. aureus can evade neutrophil responses successfully by resisting microbicides, surviving intracellularly and subverting cell death pathways. Developing a detailed understanding of these complex strategies is especially relevant in light of increasing staphylococcal virulence and antibiotic resistance, and the knowledge that dysfunctional neutrophil responses contribute materially to poor host outcomes. Unravelling the biology of these interactions is a challenging task, but one which may yield new strategies to address this, as yet, defiant organism.

Pathological networking: a new approach to understanding COPD.

Developing new treatments for chronic obstructive pulmonary disease (COPD) is extremely challenging. This disease, chronic by definition, becomes apparent only after substantial--and probably irreversible--tissue damage has occurred. The observable phenotype is of a stable disease state whose progression is hard to influence and reversal of which appears almost impossible. Identifying key components of the pathological process, targeting of which will result in substantial clinical benefit, is a significant challenge. In this review the nature of the disease is examined and conceptual information and simple tissue models of inflammation are used to explore the pathological network that is COPD. From the concept of COPD as a disease network displaying the features of contiguous immunity (in which many processes of innate and adaptive immunity are in continual dialogue and evolution), refinements are suggested to the strategies aimed at developing effective new treatments for this disease.

The role of TLR activation in inflammation.

The Toll-like receptor family was originally identified in Drosophila, where it provides important developmental and immunological signalling. In mammals, the developmental signal appears to have been lost, but the immunological defence role of these receptors has been expanded to provide broad recognition of bacterial, fungal, viral and parasitic pathogens. There is increasing evidence that these receptors go beyond the recognition of microbial molecules to sense host tissue damage. Recognition of host molecules and commensal microbes is also involved in the restoration of normal tissue architecture after injury and in maintenance of epithelial health. Recent developments in the TLR field highlight the importance of these molecules to human health and disease and demonstrate that their targeting, to boost immunity or inhibit inflammation, is both feasible and also potentially challenging.

Polymorphisms in the endothelin-1 (EDN1) are associated with asthma in two populations.

Endothelin-1 (EDN1) has been reported to be implicated in the pathophysiology of asthma. Literature results on the genetic association of EDN1 in asthma are inconsistent. Eleven single nucleotide polymorphisms in EDN1 were genotyped in 342 and 100 families from UK and Norway, respectively. Asthma, bronchial hyperreactivity (BHR) and atopic asthma phenotypes were analyzed for the family-based association. Five single nucleotide polymorphisms (SNPs) were associated with asthma (0.0017

Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation in respiratory disease.

The neutrophil is a crucial early defence against microbial infection, but neutrophilic inflammation can result in devastating acute and chronic inflammatory diseases. In the lungs, the neutrophil is a principal part of the pathology of the acute respiratory distress syndrome, and its activation may also be of substantial importance in chronic obstructive pulmonary disease and some forms of asthma. Induction of neutrophil recruitment in response to microbial attack requires activation of TLR (Toll-like receptor)-based signalling pathways and the concerted actions of multiple cell types, including sentinel cells such as monocytes and macrophages acting together with tissue cell types such as the epithelium or smooth-muscle cell. The present review describes some of these networks and the resulting potential for their targeting in respiratory disease.

Asthma severity and atopy: how clear is the relationship?

BACKGROUND: The relationship between asthma severity and atopy is complex. Many studies have failed to show significant relationships between clinical severity or lung function and markers of atopic sensitisation. AIM: To determine whether increasing asthma severity is related to atopic sensitisation in a population of children with asthma. METHODS: A total of 400 children (7-18 years) with asthma were recruited as part of a multicentre study of the genetics of asthma. Detailed phenotypic data were collected on all participants. Associations between measures of asthma severity and atopic sensitisation were sought using multilevel models allowing variation at the individual and family level. RESULTS: Children recruited to the study had a range of asthma severities, with just over a third having mild persistent asthma. The logarithm of total serum IgE was associated with increased asthma severity score, decreased FEV1, increased airways obstruction, risk of hospital admission, and inhaled steroid use. Increasing skin prick test reactivity to a panel of seven aeroallergens was associated with increased risk of hospital admission, use of an inhaled steroid, and airways obstruction. The results remained highly significant after corrections for age, gender, and birth order. CONCLUSIONS: In children with asthma, increasing atopy is associated with increasing asthma severity. However, the relationships between asthma severity and skin prick tests, and asthma severity and total serum IgE values, appear subtly different.

Effects of glutathione S-transferase M1, T1 and P1 on lung function in asthmatic families.

RATIONALE: Previous data have suggested that glutathione-S-transferase (GST) genotypes are important in determining the rate of lung function growth in childhood. This effect was most marked in Caucasian children with asthma. OBJECTIVES: We investigated the association of lung function with GSTM1, GSTP1 and GSTT1 genotypes in Caucasian families with asthma. METHODS: Four hundred and eighteen children and 316 parents from 224 Caucasian families were recruited via a child with asthma, the proband. Associations between lung function and GST genotype were determined using multilevel models. RESULTS: There were no observed associations between lung function and GST genotype in parents. However, in the children, the GSTP1 val(105)/val(105) and GSTM1 null genotypes were associated with significantly higher forced expiratory volume in 1 s (FEV(1)) and FVC values as percentage of predicted. This effect was not statistically significant in the probands but was marked in their siblings in whom GSTP1 val(105)/val(105) was associated with 9.4% higher FEV(1) and 10.7% higher FVC (P=0.005 and 0.001, respectively). The GSTM1 null genotype was associated with a 6.7% higher FEV(1) and 4.1% higher FVC (P=0.003 and 0.063, respectively). These effects remained significant after correcting for the confounders of individual atopic status, tobacco smoke exposure and familial aggregation of lung function values. CONCLUSIONS: GSTM1 and GSTP1 genotypes are important determinants of lung function in childhood. The smaller differences seen in probands are predicted by a simple model in which more rapid decline in lung function is seen in these individuals.

What can we learn from highly purified neutrophils?

Neutrophil purification has traditionally been performed by centrifugation of leucocytes through density gradients. These reliable methods produce populations that are typically >95% pure neutrophils, and have allowed the widespread study of the function of these cells. Our recent work has suggested that residual monocytes may play a more important role than has been previously realized, and suggest that for some functional experiments, further purification of cells is required to understand fully the neutrophil phenotype.

The role of neutrophil apoptosis in the resolution of acute lung injury in newborn infants.

BACKGROUND: The persistent airway neutrophilia observed in chronic lung disease of prematurity (CLD) may reflect inappropriate suppression of neutrophil apoptosis. METHODS: 134 bronchoalveolar lavage (BAL) samples were obtained from 32 infants requiring mechanical ventilation for respiratory distress syndrome (RDS): 13 infants (median gestation 26 weeks, range 23 to 28) subsequently developed CLD (CLD group), and 19 infants (gestation 31 weeks, range 25 to 39) recovered fully (RDS group). A further 73 BAL samples were obtained from 20 infants (median age 2 days, range 1 to 402) receiving extracorporeal membrane oxygenation (ECMO) for severe respiratory failure. RESULTS: Neutrophil apoptosis was increased in the RDS group (mean (SEM) neutrophil apoptosis on day 7 BAL: RDS 17.0 (8.6)% v CLD 0.7 (0.2)% (p<0.05)). BAL fluid obtained from RDS but not CLD patients was proapoptotic to neutrophils (apoptosis ratio BAL fluid/saline control: day 1, RDS 9.8 (5.5) v CLD 1.2 (0.1) (p<0.05); day 2, RDS 4.32 (2.8) v CLD 0.5 (0.4) (p<0.05)). There were similar findings in the ECMO group: survivors had proapoptotic BAL fluid compared with non-survivors (apoptosis ratio day 1, survivors 7.9 (2.1) v non-survivors 2.1 (0.7) (p<0.05)). CONCLUSIONS: Inappropriate suppression of neutrophil apoptosis may be associated with a poor outcome in newborn infants with respiratory failure.

Diagnostic and treatment behaviour in children with chronic respiratory symptoms: relationship with socioeconomic factors.

BACKGROUND: The prevalence and severity of asthma is believed to increase with increasing socioeconomic deprivation. The relationship between asthma diagnosis, symptoms, diagnostic accuracy, and socioeconomic deprivation as determined by Townsend scores was determined in Sheffield schoolchildren. METHODS: All 6021 schoolchildren aged 8-9 years in one school year in Sheffield were given a parent respondent survey based on International Survey of Asthma and Allergies in Childhood (ISAAC) questions. RESULTS: 5011/6021 (83.2%) questionnaires were returned. Postcode data were available in 4131 replies (82.4%) and were used to assign a composite deprivation score (Townsend score). Scores were divided into five quintiles, with group 1 being least and group 5 being most deprived. A positive trend was observed from group 1 to group 5 for the prevalence of wheeze in the previous 12 months, wheeze attacks >or=4/year, nocturnal wheeze and cough (all p<0.001), cough and/or wheeze "most times" with exertion (p<0.03), current asthma (p<0.001), and significant asthma symptoms (p<0.001). No significant trend was observed for lifetime wheeze or attacks of speech limiting wheeze. There were no significant trends in the prevalence of current asthmatic children without significant symptoms (overdiagnosis) or children with significant asthma symptoms but no current asthma diagnosis (underdiagnosis) across the social groups. There was a significant negative trend in the ratio of asthma medication to asthma diagnosis from least to most deprived groups (p<0.001). CONCLUSIONS: Asthma morbidity and severity increase according to the level of socioeconomic deprivation. This may be due to differences in environment, asthma management, and/or symptom reporting. Diagnostic accuracy does not vary significantly across deprivation groups but children living in areas of least deprivation and taking asthma medication are less likely to be labelled as having asthma, suggesting diagnostic labelling bias.

Toll-like receptors: their role in allergy and non-allergic inflammatory disease.

The human TLRs comprise an important and interesting group of receptors that regulate pathogen-related responses, and play as yet uncharacterized roles in the amplication of sterile inflammation. Signalling through these receptors, which are powerfully coupled in gene transcription processes, has powerful immunostimulatory and immunomodulatory effects. Exploitation of TLR signalling will probably lead to novel effective therapies for allergic disease, in the first instance through more efficient mechanisms of immunotherapy. The likelihood of adverse consequences of such treatments, though possible, may be minimized by use of conjugated vaccines.

Chemokines, innate and adaptive immunity, and respiratory disease.

Selective leukocyte trafficking and recruitment is primarily regulated by a specific family of small proteins called "chemokines". This extended family shepherds and guides leukocytes through their lives, facilitating their development, regulating their interactions with other leukocyte types, and guiding their recruitment to sites of inflammation. Through the actions of chemokines, allergen sensitization is regulated in atopic asthma, through the controlled migration of dendritic cells, T- and B-lymphocytes, mast cells and basophils. Subsequently, atopic inflammation is driven by chemokine-directed recruitment of eosinophils, basophils and lymphocytes. Diseases from cancer to chronic obstructive pulmonary disease to interstitial fibrosis are all potential targets for chemokine receptor antagonism. Innate immunity (the early pattern-recognition responses to stimuli such as lipopolysaccharide, viral proteins and bacterial DNA) needs to bridge the gap to specific immunity and antibody production and immunological memory. Again, chemokines are likely to be fundamental mediators of these responses. Chemokines are fundamental regulators of leukocyte homeostasis and inflammation, and their antagonism by small molecule chemokine receptor antagonists may be of enormous importance in the future treatment of human respiratory disease.

Increasing prevalence of asthma diagnosis and symptoms in children is confined to mild symptoms.

BACKGROUND: The prevalence of childhood asthma is increasing but few studies have investigated trends in asthma severity. We investigated trends in asthma diagnosis and symptom morbidity between an eight year time period in a paired prevalence study. METHODS: All children in one single school year aged 8-9 years in the city of Sheffield were given a parent respondent questionnaire in 1991 and 1999 based on questions from the International Survey of Asthma and Allergy in Children (ISAAC). Data were obtained regarding the prevalence of asthma and wheeze and current (12 month) prevalences of wheeze attacks, speech limiting wheeze, nocturnal cough and wheeze, and exertional symptoms. RESULTS: The response rates in 1991 and 1999 were 4580/5321 (85.3%) and 5011/6021 (83.2%), respectively. There were significant increases between the two surveys in the prevalence of asthma ever (19.9% v 29.7%, mean difference 11.9%, 95% confidence interval (CI) 10.16 to 13.57, p<0.001), current asthma (10.3% v 13.0%, mean difference 2.7%, 95% CI 1.44 to 4.03, p<0.001), wheeze ever (30.3% v 35.8%, mean difference 5.7%, 95% CI 3.76 to 7.56, p<0.001), wheeze in the previous 12 months (17.0% v 19.4%, mean difference 2.5, 95% CI 0.95 to 4.07, p<0.01), and reporting of medication use (16.9% v 20%, mean difference 3.0%, 95% CI 1.46 to 4.62, p<0.001). There were also significant increases in reported hayfever and eczema diagnoses. CONCLUSIONS: Diagnostic labelling of asthma and lifetime prevalence of wheeze has increased. The current 12 month point prevalence of wheeze has increased but this is confined to occasional symptoms. The increased medication rate may be responsible for the static prevalence of severe asthma symptoms. The significant proportion of children receiving medication but reporting no asthma symptoms identified from our 1999 survey suggests that some children are being inappropriately treated or overtreated.